Methods and compositions for the treatment of obesity, insulin related diseases and hypercholesterolemia

ABSTRACT

A process of enhancing insulin excretion in a subject includes administering to the subject a polyphenol active ingredient. The polyphenol active ingredient is a purified cyanidin-3-glycoside alone or purified cyanidin alone. The pharmaceutically acceptable carrier is administered with the polyphenol active ingredient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/072,151 filed Mar. 4, 2005, which relies for priority on ProvisionalPatent Application Ser. No. 60/591,806, filed Jul. 29, 2004.

GOVERNMENT RIGHTS

This invention was funded under USDA Grant No. 2003-35504-13618. TheU.S. Government has certain rights to this invention.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to the treatment of insulin relateddiseases, obesity, diabetes mellitus, hyperglycemia, lipid disorders,hyperlipidemia, or low HDL, hypercholesterolemia, hyperglyceridemia,dyslipidemia, and atherosclerosis. The present invention particularlyrelates to a method which uses anthocyanins, anthocyanidins, ursolicacid and betulinic acid. The invention particularly relates to Cornusspp. fruit extracts as well as other fruits containing these compounds,such as cherries and berries, or mixtures thereof to increase insulinproduction by cells in vivo. The present invention also particularlyrelates to compositions to be used in the method for producing theincrease in production of the insulin in vivo in the treatment of therelated diseases. The present invention also particularly relates tocompositions used to prevent obesity and lowering cholesterol and bodyweight.

(2) Description of the Related Art

The function of insulin is to maintain normal blood glucose levelseither by suppression of glucose output from liver or by the stimulationof glucose uptake and its metabolism (Ross, S. A.; Gulve, E. A.; Wang,M. Chemistry and Biochemistry of diabetes. Chem. Rev. 2004, 104,1255-1282). Insufficient release of insulin or loss of insulin action attarget tissues causes aberrant glucose and lipid metabolism. Thisresults in elevated glucose levels in the blood, a hallmark of diabetes(Jovanovic, L.; Gondos, B. Type-2 diabetes: The epidemic of newmillennium. Ann. Clin. Lab. Sci. 1999, 29, 33-42). There are two typesof diabetes, type-1 (insulin-dependent diabetes) and type-2 diabetes(non-insulin-dependent diabetes). Type-1 diabetes results fromautoimmune destruction or inhibition of pancreatic β-cells, the cellsthat secrete insulin, which leads into insulin insufficiency. Type-2diabetes is more prevalent and is caused by the inability of β-cells tosecrete sufficient amounts of insulin to overcome insulin resistanceestablished by genetic and environmental factors (Henquin, J. C.Triggering and amplifying pathways of regulation of insulin secretion byglucose. Diabetes 2000, 49, 1751-1760). The insulin resistance is adisorder in which insulin inadequately stimulates glucose transport inskeletal muscle and fat and inadequately suppresses hepatic glucoseproduction. The mechanisms involved that prevent the β-cell fromsecreting sufficient amounts of insulin to overcome peripheral insulinresistance remain to be established.

Oral hypoglycemic agents that directly stimulate insulin release fromβ-cells (e.g. sulfonylurea based drugs as exemplified by U.S. Pat. No.6,852,738 to Jones et al., incorporated herein by reference), however,have shown that insulin secretion from islets of type-2 diabeticpatients can be elevated sufficiently to overcome peripheral insulinresistance and normalize blood glucose levels. One of the disadvantagesof using sulfonylurea-based drugs is that they fail to control normalblood glucose levels (Pfeiffer, A. F. H. Oral hypoglycemic agents:Sulfonylureas and meglitinides. In B. J. Goldstein, D. Muller-Wieland(Eds.), Text book of Type-2 Diabetes. Martin Dunitz Ltd., London, 2003,pp. 77-85). These drugs also adversely affect the ability of β-cells tosecrete insulin and cause weight gain (Pfeiffer, A. F. H. Oralhypoglycemic agents: Sulfonylureas and meglitinides. In B. J. Goldstein,D. Müller-Wieland (Eds.), Text book of Type-2 Diabetes. Martin DunitzLtd., London, 2003, pp. 77-85). Hence, there is a role for dietaryconstituents that can regulate blood glucose level or induce insulinproduction by pancreatic β-cell in addition to traditional ethical drugtreatment.

Reports indicate that consumption of fruits and vegetables, especiallyrich in polyphenols, decreased the incidence of type-2 diabetes(Anderson, R. A.; Polansky, M. M., Tea Enhanced Insulin Activity. J.Agric. Food Chem. 2002, 50, 7182-7186; Anderson, R. A.; Broadhurst, C.L.; Polansky, M. M.; Schmidt, W. F.; Khan, A.; Flanagan, V. P.; Schoene,N. W.; Graves, D. J. Isolation and Characterization of Polyphenol Type-APolymers from Cinnamon with Insulin-like Biological Activity. J. Agric.Food Chem. 2004, 52, 65-70; Landrault, N.; Poucheret, P.; Azay, J.;Krosniak, M.; Gasc, F.; Jenin, C.; Cros, G.; Teissedre, P. Effect of aPolyphenols-Enriched Chardonnay White Wine in Diabetic Rats. J. Agric.Food Chem. 2003, 51, 311-318). Also, it is known that dietaryantioxidants protect pancreatic β-cells from glucose-induced oxidativestress. Anthocyanins are abundant in fruits, vegetables and processedfood products such as wine, cider and tea. However, little is known ofits ability to reduce or prevent diabetes.

Also, anthocyanins are nontoxic and reported to possess antioxidant,anti-inflammatory and anticancer activities (Wang, H., Nair, M. G.,Strasburg, G. M., Chang, Y., Booren, A. M. Gray, J. I., and DeWitt, D.L. (1999) Antioxidant and anti-inflammatory activities of anthocyaninsand their aglycon, cyanidin, from tart cherries. J. Nat. Prod. 62,294-296; Tall, J. M., Seeram, N. P., Zhao, C., Nair, M. G., Meyer, R.A., and Raja, S. N. (2004) Tart cherry anthocyanins suppressinflammation-induced pain behavior in rat. Behav. Brain Res. 153,181-188; Kang, S., Seeram, N. P., Nair, M. G., and Bourquin, L. D.(2003) Tart cherry anthocyanins inhibit tumor development in ApcMin miceand reduce proliferation of human colon cancer cells. Canc. Lett. 194,13-19; Zhang, Y., Vareed, S. K., and Nair, M. G. (2005) Human tumor cellgrowth inhibition by nontoxic anthocyanidins, the pigments in fruits andvegetables. Life Sci. 76, 1465-1472.).

The bioactive natural products present in vegetables, fruits and herbshave generated considerable interest in prevention and treatment ofhuman degenerative disorders like cancer, diabetes and cardiovasculardiseases. For example, nuts, whole grains, fruits, and vegetables arerich source of antioxidants such as polyphenols, terpenoids and pigmentsand these compounds have been associated with the amelioration ofseveral disease conditions. Similarly, phytochemicals present in garlic,soybeans, cabbage, ginger, licorice, and the umbelliferous vegetablesare known to possess anticancer activity (Rui, H. L. (2004) Potentialsynergy of phytochemicals in cancer prevention: mechanism of action. J.Nutr. 134, 3479S-3485S). Also, the polyphenols present in tea arereported to possess anti-diabetic properties (Vanessa, C., and Gary, W.(2004) A review of the health effects of green tea catechins in in vivoanimal models. J. Nutr. 134, 3431S-3440S and Mary E. W., Xiaohui, L. W.,Brian, K. L., Robert K. H., Masao, N., and Daryl K. G. (2002)Epigallocatechin gallate, a constituent of green tea, represses hepaticglucose production. J. Biol. Chem. 277, 34933-34940.).

The consumption of a diet low in fat and rich in antioxidants reducesthe risk of obesity and insulin resistance (Blakely, S.; Herbert, A.;Collins, M.; Jenkins, M.; Mitchell, G.; Grundel, E.; O'Neill, K. R.;Khachik, F. Lutein interacts with ascorbic acid more frequently thanwith α-tocopherol to alter biomarkers of oxidative stress in femaleZucker obese rats. J. Nutr. 2003, 133, 2838-2844).

Anthocyanins belong to antioxidant polyphenols and are present invarious foods and beverages. Consumption of anthocyanins is associatedwith reduced risk of several degenerative diseases such asatherosclerosis, cardiovascular disease, cancer and diabetes(Jayaprakasam, B.; Strasburg, G. A.; Nair, M. G. Potent lipidperoxidation inhibitors from Withania somnifera, Tetrahedron 2004, 60,3109-3121). These compounds are well-known free radical scavengers andreported as potential chemopreventive agents (Duthie, G. G.; Duthie, S.J.; Kyle, J. A. M. Plant polyphenols in cancer and heart disease:implications as nutritional antioxidants. Nutr. Res. Rev. 2000, 13,79-106). For example, serum antioxidant capacity was increased by theconsumption of strawberries, cherries, and red wine (Kang, S. Y.;Seeram, N. P.; Nair, M. G.; Bourquin, L. D. Tart cherry anthocyaninsinhibit tumor development in ApcMin mice and reduce proliferation ofhuman colon cancer cells. Canc. Lett. 2003, 194, 13-19; Van Velden, D.P.; Mansvelt, E. P. G.; Fourie, E.; Rossouw, M.; Marais, A. D. Thecardioprotective effect of wine on human blood chemistry. Ann. New YorkAcad. Sci. 2002, 957, 337-340; Wang, H.; Nair, M. G.; Strasburg, G. M.;Chang, Y. C.; Booren, A. M.; Gray, I. J.; DeWitt, D. L. Antioxidant andanti-inflammatory activities of anthocyanins and their aglycone,cyanidin, from tart cherries. J. Nat. Prod. 1999, 62, 294-296). Recentstudies demonstrated that the anthocyanin, cyanidin 3-glucoside, reducedthe high fat diet induced obesity in mice (Espin, J. C.; Soler-Rivas,C.; Withers, H. J.; Garcia-Viguera, C. Anthocyanin-based naturalcolorants. A new source of antiradical activity for foodstuff. J. Agri.Food Chem. 2000, 48, 1588-1592). Therefore, the natural colorantspresent in the food have attracted consumers due to their safety,nutritional and therapeutic values (Millspaugh, C. F. In AmericanMedicinal Plants; Dover Publications: New York, 1974; p 282). Sinceanthocyanins are widely consumed, additional biological activities ofthese compounds are of great interest.

Several studies suggest that diets rich in fat and low in fiber resultin obesity. Obesity alters the lipid metabolism, which in turn leads toinsulin resistance. Under obese conditions, the adipose tissue producesan enormous amount of free fatty acids (FFA). The FFA then inhibits theglucose uptake, glycogen synthesis and glucose oxidation (Saltiel, A. R.and Kahn, C. R. (2001) Insulin signaling and the regulation of glucoseand lipid metabolism. Nature 414, 799-806) and results in hyperglycemiaand type-2 diabetes. The type-2 diabetes is an increasingly commondisorder and approximately 150 to 300 million people suffer worldwideand expected to double in the next 25 years (King, H., Aubert, R. E.,and Herman, W. H. (1998) Global burden of diabetes, 1995-2025:prevalence, numerical estimates, and projections. Diabetes Care 21,1414-1431). Recently, much attention has been focused on food that maybe beneficial in preventing diet-induced body fat accumulation andpossibly reduce the risk of diabetes and heart disease.

There are several biochemical processes involved in controlling the foodintake. The glucagons-like-peptide-1 and -2 (GLP-1 & -2) weresynthesized in endocrine cells and released into the blood in responseto nutrients intake. The GLP-2 enhances the nutrient absorption byexpanding the mucosal epithelium (Ahren, B. (1998) Glucagon-likepeptide-1 (GLP-1): a gut hormone of potential interest in the treatmentof diabetes. BioEssays 20, 642-651 and Drucker, D. J. (2002) Biologicalaction and therapeutic potential of glucagons like peptides.Gastroenterology 122, 531-544). The GLP-1 is mainly expressed in gutL-cells and it inhibits glucagon secretion and gastric emptying by theliver, which in turn inhibit the food intake and stimulates insulinbiosynthesis and secretion by pancreatic β-cells (Ahren, B. (1998)Glucagon-like peptide-1 (GLP-1): a gut hormone of potential interest inthe treatment of diabetes. BioEssays 20, 642-651 and Drucker, D. J.(2002) Biological action and therapeutic potential of glucagons likepeptides. Gastroenterology 122, 531-544). The primary function ofpancreatic β-cells is to secrete the bioactive insulin, in response tonutrients, hormones and nervous stimuli, in order to keep the normalphysiological glucose concentrations of the body (Rohit, N. K. (2004)The islet β-cell. The Int. J. Biochem. Cell Biol. 36, 365-371). Theprogressive loss of the pancreatic β-cell function in response toelevated blood glucose levels causes the insulin deficiency, which leadsto type-2 diabetes. The insulin resistance, the failure of liver,muscle, and adipose tissue to respond to physiologic doses of insulin,also causes type-2 diabetes (Pinget, M., and Boullu-Sanchis, S. (2002)Physiological basis of insulin secretion abnormalities. Diabet. Met. 28(6, Suppl.), 4S21-4S32). Both insulin deficiency and resistance lead tohealth problems such as hyperlipidemia, atherosclerosis and hypertension(Saltiel, A. R. and Kahn, C. R. (2001) Insulin signaling and theregulation of glucose and lipid metabolism. Nature 414, 799-806) andoften linked to the impaired carbohydrate and lipid metabolism (Brosche,T. (2001) Plasmalogen levels in serum from patients with impairedcarbohydrate or lipid metabolism and in elderly subjects with normalmetabolic values. Arch. Gerontol. Geriatrics 32, 283-294). These controlsystems interact in complex pathways and any alteration by genetic,environmental and social factors causes obesity and diabetes (Ross, S.A., Gulve, E. A., and Wang, M. (2004) Chemistry and biochemistry of type2 diabetes. Chem. Rev, 104, 1255-1282). However, some of thecomplications resulting from social and environmental factors may bedelayed or prevented by exercise and proper diet (Christian, K. R., andBarnard, R. J. (2005) Effects of exercise and diet on chronic disease.J. Appl. Physiol. 98, 3-30). Epidemiological studies showed that dietsrich in fruits and vegetables reduce the incidence of cancer,cardiovascular disease, diabetes, cataracts, and inflammatory disease(World Cancer Research Fund/American Institute for Cancer Research(1997) Food, nutrition and the prevention of cancer: A globalperspective 1997, American Institute for Cancer Research Washington,D.C.; U.S. Department of Agriculture, U.S. Department of Health andHuman Services (1995) Nutrition and Your Health: Dietary Guidelines forAmericans 1995, U.S. Government Printing Office Washington, D.C.;American Heart Association (1996) Dietary guidelines for healthyAmerican adults: A statement for health professionals from the nutritioncommittee, American Heart Association. Circulation 94, 1795-1800;American Cancer Society (1996) Guidelines on diet, nutrition, and cancerprevention: reducing the risk of cancer with healthy food choices andphysical activity. Cancer J. Clin. 46, 325-341; World HealthOrganization (1990) Diet, Nutrition and the prevention of chronicdiseases: Report of a WHO study group, Technical Report Series 797, WHOGeneva, Switzerland; Willett, W. C. (1999) Goals for nutrition in theyear 2000. Cancer J. Clin. 49, 331-352 and Willett, W. C. (1998)Nutritional Epidemiology 1998, Press: Oxford University, New York, N.Y.,USA).

Recently, there has been an increased interest in natural hypoglycemiccompounds derived from generally regarded as safe (GRAS) plants, fruitsand vegetables since they are considered to be less toxic with fewerside effects. These bioactive compounds present in the food can altergene expression and cellular events (Milner, J. A. (2004) Moleculartargets for bioactive food components. J. Nutr. 134, 2492S-2498S)resulting in the modification of proteins and their functions. Althoughseveral studies suggested that the phytochemicals present in the fruitsand vegetables are beneficial to ameliorate adverse health risks, theiranecdotal protective effects have not been well understood.

The Cornus fruits are used in anti-diabetic traditional Chineseprescription medicines such as “Hachimi-Gan” (Yamahara, J.; Mibu, H.;Sawada, T.; Fujimura, H.; Takino, S.; Yoshikawa, M.; Kitagawa, I.Biologically active principles of crude drugs. Anti-diabetic principlesof corni fructus in experimental diabetes induced by streptozotocin.Yakugaku Zasshi 1981, 101, 86-90). We have recently reported thequantification of anthocyanins in Cornus spp. fruits (Seeram, N. P.;Schutzki, R.; Chandra, A.; Nair, M. G. Characterization, Quantification,and Bioactivities of Anthocyanins in Cornus Species. J. Agri. Food Chem.2002, 50, 2519-2523).

The fruits of the Cornus species are a rich source of anthocyanins. Thefruits of Cornus mas L., also known as the European and AsiaticCornelian cherry, are used in the preparation of beverages in Europe(Kim, D. K.; Kwak, J. H. A Furan derivative from Cornus officinalis.Arch. Pharm. Res. 1998, 21, 787-789). In traditional medicine, Cornusofficinalis fruits are known for their analgesic and diuretic activities(Yamahara, J.; Mibu, H.; Sawada, T.; Fujimura, H.; Takino, S.;Yoshikawa, M.; Kitagawa, I. Biologically active principles of crudedrugs. Anti-diabetic principles of carni fructus in experimentaldiabetes induced by streptozotocin. Yakugaku Zasshi 1981, 101, 86-90).The Cornus fruits are also one of the major constituents of severalanti-diabetic herbal preparations in Asian countries (Seeram, N. P.;Schutzki, R.; Chandra, A.; Nair, M. G. Characterization, Quantification,and Bioactivities of Anthocyanins in Cornus Species. J. Agri. Food Chem.2002, 50, 2519-2523). Earlier investigation of the fruits of C. mas andC. officinalis revealed that both contained high levels of anthocyanins(Beckwith, A. G.; Zhang, Y.; Seeram, N. P.; Cameron, A. C.; Nair, M. G.Relationship of Light Quantity and Anthocyanin Production in Pennisetwnsetaceum Cvs. Rubrum and Red Riding Hood. J. Agric. Food Chem. 2004, 52,456-461).

The C. mas plant yields fruits similar to tart cherry (P. cerasus). Itbelongs to the family Cornaceae and is a deciduous tree native to Europeand western Asia (Millspaugh, C. F. American Medicinal Plants; DoverPublications: New York, 1974; p 282). The fruits of this species havebeen used in Turkey to make several concoctions. Earlier studiesdemonstrated that the alcoholic extract of Cornus officinalis increasedthe GLUT 4 mRNA expression, a glucose transporter, in non-insulindependent diabetes mellitus (NIDDM) rats (Qian, D., Zhu, Y., and Zhu, Q.(2001) Effect of alcohol extract of Cornus officinalis Sieb. et Zucc onGLUT4 expression in skeletal muscle in type 2 (non-insulin-dependent)diabetes mellitus rats. Zhongguo Zhongyao Zazhi 26, 859-862). The Cornusfruits are well known in the Chinese medicine as well and the fruits ofthis species are used to cure diabetes in China. However, the activecompounds that are responsible for the anti-diabetic activity have notbeen characterized. Earlier studies on fruits from several Cornus spp.grown in Michigan revealed that C. mas contained high levels ofanthocyanins (Seeram, N. P., Schutzki, R., Chandra, A., and Nair, M. G.(2002) Characterization, quantification, and bioactivities ofanthocyanins in Cornus species. J. Agric. Food Chem. 50, 2519-2523).

OBJECTS

It is an object of the present invention to provide compositions andmethods which are useful in the treatment of disorders related toinsulin production, high cholesterol and body weight. In particular itis an object of the present invention to provide a method andcompositions for increasing insulin production in vivo. Further objectswill become apparent from the following description and the drawings.

SUMMARY OF THE INVENTION

The present invention relates to a method for controlling obesity in amammalian patient in need of such treatment which comprisesadministering to the patient a therapeutically effective amount ofcomposition comprising a compound selected from the group consisting ofan anthocyanin, an anthocyanidin, ursolic acid, betulinic acid, andmixtures thereof. The present invention particularly relates tosupplement from Cornus and other fruits which is substantially free ofacids and sugars naturally present in the fruits.

The present invention also relates to a method for treating obesity in ahuman patient who is diabetic and being treated with a prescription drugfor the diabetes which comprises administering to the patient aneffective amount of a composition comprising a compound selected fromthe group consisting of an anthocyanin, an anthocyanidin, ursolic acid,betulinic acid, and mixtures thereof in conjunction with theprescription drug. Preferably the composition is a supplement orpharmaceutical formulation.

Preferably the anthocyanin, anthocyanidin, ursolic acid or betulinicacid are isolated from fruits, vegetables and flowers. Preferably theanthocyanin is selected from the group consisting ofcyanidin-3-glycoside, delphindin-3-glycoside, pelargonidin-3-glycosideand mixtures thereof. A “glycoside” is any compound that contains acarbohydrate molecule (sugar), particularly any such natural product inplants, convertible, by hydrolytic cleavage, into sugar and a nonsugarcomponent (aglycone), and named specifically for the sugar contained, asglucoside (glucose), pentoside (pentose), fructoside (fructose), etc.Preferably the anthocyanin, ursolic acid or betulinic acid are isolatedfrom Cornus mas. Preferably the anthocyanidin, or anthocyanin,anthocyanidin, ursolic acid, betulinic acid or mixtures thereof areisolated and purified.

The present invention also relates to a method for the treatment ofdiabetes mellitus in a mammalian patient in need of such treatment forcontrolling the diabetes mellitus which comprises administering to saidpatient a therapeutically effective amount of a composition comprising acompound selected from the group consisting of an anthocyanin, ananthocyanidin, ursolic acid, betulinic acid or mixtures thereof.

Preferably the anthocyanin, ursolic acid or betulinic acid are isolatedfrom fruits, vegetables and flowers. Preferably the anthocyanin isselected from the group consisting of cyanidin-3-glycoside,delphinidin-3-glycoside, pelargonidin-3-glycoside and mixtures thereof.Preferably the anthocyanin, ursolic acid, or betulinic acid are isolatedfrom Cornus mas. However, anthocyanins can be from other fruits such ascherries and berries.

The present invention also relates to a method for treating orcontrolling hyperglycemia in a mammalian patient in need of suchtreatment which comprises administering a therapeutically effectiveamount of a composition comprising a compound selected from the groupconsisting of an anthocyanin, ursolic acid, betulinic acid and mixturesthereof, particularly as a supplement.

Preferably the anthocyanin, anthocyanidin, ursolic acid or betulinicacid are isolated from fruits, vegetables and flowers. Preferably theanthocyanin is selected from the group consisting ofcyanidin-3-glycoside, delphinidin-3-glycoside, pelargonidin-3-glycosideand mixtures thereof. Preferably the anthocyanin, ursolic acid orbetulinic acid are isolated from Cornus mas. Preferably theanthocyanidin, anthocyanin, ursolic acid, betulinic acid or mixturesthereof are isolated and purified.

The present invention also relates to a composition for use in treatmentof obesity, diabetes, or hyperglycemia as a disease which comprises: ananthocyanin, anthocyanidin, ursolic acid or betulinic acid or mixturesthereof in a daily dosage unit for treatment of the disease over aperiod of time; and a pharmaceutical carrier.

Most preferably the anthocyanin is selected from the group consisting ofcyanidin-3-glucoside, delphinidin-3-glucoside, pelargonidin-3-glucosideand mixtures thereof. Preferably the anthocyanin, anthocyanidin, ursolicacid or betulinic acid are isolated from fruits, vegetables or flowers.Preferably the anthocyanin, ursolic acid or betulinic acid are isolatedfrom Cornus mas.

The present invention also relates to a method for treating orcontrolling lipid disorders, hyperlipidemia, or low HDL in a mammalianpatient in need thereof which comprises administering to said patient atherapeutically effective amount of a composition comprising a compoundconsisting of an anthocyanin, anthocyanidin, ursolic acid, betulinicacid, or mixtures thereof, particularly as a supplement.

The present invention also relates to a method for treating orcontrolling hypercholesterolemia in a mammalian patient in need of suchtreatment which comprises administering to said patient atherapeutically effective amount of a composition comprising a compoundconsisting of an isolated anthocyanin, anthocyanidin, ursolic acid,betulinic acid, or mixtures thereof, particularly as a supplement.

The present invention also relates to a method for treating orcontrolling hyperglyceridemia in a mammalian patient in need of suchtreatment which comprises administering to said patient atherapeutically effective amount of a composition comprising a compoundselected from the group consisting of an isolated anthocyanin,anthocyanidin, ursolic acid, betulinic acid, or mixtures thereof,particularly as a supplement.

The present invention also relates to a method for treating orcontrolling dyslipidemia and/or low HDL cholesterol in a mammalianpatient in need of such treatment which comprises administering to saidpatient a therapeutically effective amount of a composition comprising acompound selected from the group consisting of an isolated anthocyanin,anthocyanidin, ursolic acid, betulinic acid, or mixtures thereof,particularly as a supplement.

The present invention also relates to a method for treatment ofatherosclerosis in a mammalian patient in need of such treatment whichcomprises administering to said patient a therapeutically effectiveamount of a composition comprising a compound selected from the groupconsisting of an isolated anthocyanin, anthocyanidin, ursolic acid,betulinic acid, or mixtures thereof, particularly as a supplement.

Most preferably the compound is from Cornus mas. Preferably theanthocyanin, anthocyanidin, ursolic acid, betulinic acid or mixturesthereof are isolated and purified from this or other fruits.

Metabolites of the compounds of this invention that are therapeuticallyactive are within the scope of the claimed parent compounds. Prodrugs,which are compounds that are converted to the claimed compounds as theyare being administered to a patient or after they have been administeredto a patient, are also within the scope of the claimed active compounds.

Administration and Dose Ranges

Any suitable route of administration may be employed for providing amammal, especially a human, with an effective dose of a compound of thepresent invention. For example, oral, rectal, topical, parenteral,ocular, pulmonary, nasal, and the like may be employed. Dosage formsinclude tablets, troches, dispersions, suspensions, solutions, capsules,creams, ointments, aerosols, and the like. Preferably compounds areadministered orally.

The effective dosage of active ingredient employed may vary depending anthe particular compound employed, the mode of administration, thecondition being treated and the severity of the condition being treated.Such dosage may be ascertained readily by a person skilled in the art.

When treating or preventing obesity, diabetes mellitus and/orhyperglycemia or hypertriglyceridemia or other diseases, generallysatisfactory results are obtained when the compounds of the presentinvention are administrated at a daily dosage of from about 0.1milligram to about 100 milligram per kilogram of animal body weight,preferably given as a single daily dose or in divided doses two to sixtimes a day, or in sustained release form. For most large mammals, thetotal daily dosage is from about 1.0 milligrams to about 1000milligrams, preferably from about 1 milligram to about 50 milligrams. Inthe case of a 70 kg adult human, the total daily dose will generally befrom about 7 milligrams to about 350 milligrams. This dosage regimen maybe adjusted to provide the optimal therapeutic response.

Another aspect of the present invention provides pharmaceuticalcompositions and a pharmaceutically acceptable carrier. Thepharmaceutical compositions of the present invention comprise theclaimed compounds or pharmaceutically acceptable salt or prodrug thereofas an active ingredient, as well as a pharmaceutically acceptablecarrier and optionally other therapeutic ingredients. The term“pharmaceutically acceptable salts” refers to salts prepared frompharmaceutically acceptable non-toxic bases or acids including inorganicbases or acids and organic bases or acids.

The compositions include compositions suitable for oral, rectal,topical, parenteral (including subcutaneous, intramuscular, andintravenous), ocular (ophthalmic), pulmonary (nasal or buccalinhalation), or nasal administration, although the most suitable routein any given case will depend on the nature and severity of theconditions being treated and on the nature of the active ingredient.They may be conveniently presented in unit dosage form and prepared byany of the methods well known in the art of pharmacy.

In practical use, the compounds can be combined as the active ingredientin intimate admixture with a pharmaceutical carrier according toconventional pharmaceutical compounding techniques. The carrier may takea wide variety of forms depending on the form of preparation desired foradministration, e.g., oral or parenteral (including intravenous). Inpreparing the compositions for oral dosage form, any of the usualpharmaceutical media may be employed, such as, for example, water,glycols, oils, alcohols, flavoring agents, preservatives, coloringagents and the like in the case of oral liquid preparations, such as,for example, suspensions, elixirs and solutions, or carriers such asstarches, sugars, microcrystalline cellulose, diluents, granulatingagents, lubricants, binders, disintegrating agents and the like in thecase of oral solid preparations such as, for example, powders, bard andsoft capsules and tablets, with the solid oral preparations beingpreferred over the liquid preparations.

Because of their case of administration, tablets and capsules representthe most advantageous oral dosage unit form in which case solidpharmaceutical carriers are obviously employed. If desired, tablets maybe coated by standard aqueous or nonaqueous techniques. Suchcompositions and preparations should contain at least 0.1 percent ofactive compound. The percentage of active compound in these compositionsmay, of course, be varied and may conveniently be between about 2percent to about 60 percent of the weight of the unit. The amount ofactive compound in such therapeutically useful compositions is such thatan effective dosage will be obtained. The active compounds can also beadministered intranasally as, for example, liquid drops or spray.

The tablets, pills, capsules, and the like may also contain a bindersuch as gum tragacanth, acacia, corn starch or gelatin; excipients suchas dicalcium phosphate; a disintegrating agent such as corn starch,potato starch, alginic acid; a lubricant such as magnesium stearate; anda sweetening agent such as maltodextrin, lactose or saccharin. When adosage unit form is a capsule, it may contain, in addition to materialsof the above type, a liquid carrier such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar or both. A syrup or elixir may contain, in additionto the active ingredient, sucrose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor.

Compounds can also be administered parenterally. Solutions orsuspensions of these active compounds can be prepared in water suitablymixed with a surfactant such as hydroxyl-propylcellulose. Dispersionscan also be prepared in glycerol, liquid polyethylene glycols andmixtures thereof in oils. Under ordinary conditions of storage and use,these preparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exits. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g. glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

Compounds can be used in combination with other drugs that may also beuseful in the treatment, prevention, suppression or amelioration of thediseases or conditions. Such other drugs may be administered, by a routeand in an amount commonly used therefor, contemporaneously orsequentially with a compound. When a compound is used contemporaneouslywith one or more other drugs, a pharmaceutical composition in unitdosage form containing such other drugs and the compound is preferred.However, the combination therapy also includes therapies in which thecompound of Formula I and one or more other drugs are administered ondifferent overlapping schedules. It is also contemplated that when usedin combination with one or more other active ingredients, the compoundof the present invention and the other active ingredients may be used inlower doses than when each is used singly. Accordingly, thepharmaceutical compositions of the present invention include those thatcontain one or more other active ingredients, in addition to a compound.

DESCRIPTION OF FIGURES

FIG. 1 is a drawing showing structures of anthocyanins 1-4 andanthocyanidins 5-9.

FIG. 2A is a graph showing the amount of insulin secreted per milligramof protein by compounds 1 and 2 and FIG. 2B by compounds 5 and 6 in thepresence of 4 and 10 mM glucose. The final DMSO concentration in theassay wells was 0.1%. The results represented are the average of threeor five independent experiments and each sample was assayed induplicate. Insulin secretion by compounds 1, 2, 5 and 6 were significantat * (95% or p≦0.05) or ** (99% or p≦0.01) as determined by LSD usingthe t-test.

FIG. 3 is a graph showing the insulin secreted by compounds 3, 7-9 at 4and 10 mM glucose concentrations. The amount of insulin secreted wasnormalized to milligram protein. The final DMSO concentration in theassay wells was 0.1%. The results represented are the average of threeindependent experiments and each sample was assayed in duplicate.Insulin secretion by compounds 3, 7-9 was significant at * (95% orp≦0.05) as determined by LSD using the t-test.

FIG. 4 is a graph showing the food intake (in g) of animals during the12-week study period for obesity. Values are mean±SEM, n=8. Food intakewas measured every day and averaged for every week. There was nosignificant difference among the high fat (HF, 60% K. Cal of fat)control and treatment groups. Treatment groups received high fat dietfor 4 weeks before they were switched to diet mixed with test compounds,anthocyanins (1.0 g/Kg) betulinic and ursolic acids (0.5 g/Kg each) ofhigh fat diet. The normal diet contained 10% K. Cal.

FIGS. 5A, 5B and 5C are graphs showing body weight variation of C57BL/6J mice in obesity studies during 12 weeks of feeding. The normal andhigh fat diet controls received 10% and 60% K. Cal., respectively, intheir diet throughout the experiment. Anthocyanins, betulinic acid andursolic acid were separately mixed in high fat diet at 1.0, 0.5 and 0.5g per Kg of food, respectively. The groups treated with the compoundswere initially fed with high fat diet (60% K. Cal.) for four weeks andthen were switched to diet containing the appropriate treatment. Datarepresents mean±SEM, n=8.

FIG. 6 is a graph showing results of Glucose Tolerance Test in obesitystudies over a period of 90 min after the glucose load. The test wasconducted during the 11^(th) the week of feeding. A solution of glucose(2 g/Kg body weight) in water was administered intraperitoneally and theblood glucose level was measured at 0, 5, 15, 30, 60, and 90 min. Theblood was collected from tail vein. Vertical bars represents S.E. ateach data point n=5.

FIG. 7 is a graph showing plasma insulin levels of C57BL/6J micedetermined at the end of the feeding experiment. The plasma insulinconcentration determined for low and high fat fed control groups were0.47±0.14 and 0.41±0.1 ng/mL, respectively. The quantification ofinsulin in plasma was carried out by Radio Immuno Assay (RIA). Eachsample was assayed in duplicates and values represent mean±SEM for n=8.

FIG. 8 is a graph showing plasma cholesterol level of mice in the plasmacollected at the end of the feeding study and represented in mg/dL. Thecholesterol level for ursolic acid treated animals was not tested due toinsufficient amount of plasma sample. The values represent mean±SEM forn=4 or 5.

FIG. 9 shows the compounds isolated from 10 to 12 Cornus mas L.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present discloses and demonstrates the ability of anthocyanins,cyanidin-3-glucoside, delphinidin-3-glucoside, cyanidin-3-galactoside,and pelargonidin-3-galactoside; and anthocyanidins, cyanidin,delphinidin, pelargonidin, malvidin, and petunidin to stimulate insulinsecretion by rodent pancreatic beta cells (INS-1 813/32) in vitro. Thecompounds were tested in the presence of 4 and 10 mM glucoseconcentrations. Cyanidin-3-glucoside and delphinidin-3-glucoside werethe most effective insulin secretagogues among the anthocyanins andanthocyanidins tested at 4 and 10 mM glucose concentrations.Pelargonidin-3-galactoside is one of the major anthocyanins and itsaglycone, pelargonidin, caused a 1.4-fold increase in insulin secretionat 4 mM glucose concentration. Remaining of the anthocyanins andanthocyanidins tested had only marginal affects on insulin at 4 and 10mM glucose concentrations.

Examples of Insulin Stimulation

Materials and Methods

Chemicals. Fetal bovine serum (PBS) and RPMI-1640 medium were obtainedfrom Invitrogen (Grand Island, N.Y.). All organic solvents used were ACSreagent grade. HEPES, penicillin-streptomycin, glutamine, sodiumpyruvate, 2-mercaptoethanol, trypsin-EDTA, BSA (Bovine, Albumin; RIAGrade), Folin-Ciolatues reagent and chemicals used for the preparationof buffers were purchased from Sigma-Aldrich Chemical Co. (St. Louis,Mo.). The anthocyanidins, cyanidin, delphinidin, pelargonidin, malvidin,and petunidin, used in the assay were purchased from Chromadex (LagunaHills, Calif.).

Anthocyanins. Delphinidin-3-glucoside was purified from C. officinalisfruits. Cyanidin-3-galactoside and pelargonidin-3-galactoside wereisolated from C. mas fruits. Pure cyanidin-3-glucoside used in thisstudy was from our storage at −20° C.

Isolation and purification of anthocyanins. The Cornus fruits wereblended with water (pH=3) and filtered. The filtrate was passed throughXAD-16 AMBERLITE resin in a column and the resin with the adsorbedanthocyanins was washed repeatedly with water. The XAD-16 resin was theneluted with acidic MeOH (pH=3) and the resulting solution wasconcentrated under reduced pressure to yield a crude anthocyaninfraction. This fraction was purified by MPLC column (C18 silica) usingMeOH:H₂O (pH=3) under gradient conditions. The anthocyanins were elutedwith MeOH:H₂O (65:35, v/v) solvent system. The purity of the compoundswas checked by HPLC (Waters Corp.) using Capcell C₁₈ analytical columnunder gradient conditions. The solvents used were A:TFA:H₂O (99.9:0.1;v/v) and B: H₂O:CH₃CN:CH₃COOH:TFA (50.4:48.5:1.0:0.1; v/v/v/v). Thegradient was 20% B to 60% B in 26 min and to 20% B in 30 min at a flowrate of 0.8 ml/min. The peaks were detected at 520 nm using a PDA.

Insulin Secretion Studies. INS-1 832/13 cells (kindly provided by DrChristopher Newgard, Duke University, N.C.) were routinely cultured in5% CO₂/air at 37° C. in RPMI-1640 medium containing 11.1 mM glucose andsupplemented with 10% FBS (Fetal Bovine Serum), 10 mM HEPES, 100 U/mlpenicillin, 100 μg/ml streptomycin, 4 mM Glutamine, 1 mM sodiumpyruvate, and 50 μM 2-mercaptoethanol. Cells were passed weekly aftertrypsin-EDTA detachment. For static secretion studies, cells were platedon 24 well plates at a density of 0.64×10⁶ cells per well and grown for24 h. The cells were then cultured for an additional 24 h in RPMI-1640containing 4 mM glucose and the supplements described above. Cells werethen incubated twice for 30 min in Krebs Ringer Bicarbonate buffer(KRBB) containing 4 mM glucose and 0.1% BSA. Cells were rapidly washedwith KRBB and incubated for 60 min KRBB containing 4 or 10 mM glucosewith or without the indicated anthocyanins or anthocyanidins. The mediumwas then removed for determining insulin release. The cells were thenwashed twice with PBS and dissolved in 1 M NaOH. Cellular proteinconcentration was then determined by Lowry assay. Anthocyanins andanthocyanidins were dissolved in DMSO to obtain desired concentrations.Final concentration of DMSO was 0.1%. The insulin secreted into themedium by the cells was determined by radioimmuno assay and normalizedto total cellular protein.

Radio Immuno Assay (RIA). The Kit for RIA was purchased from LINCOResearch Inc. (St Charles, Mo.), and the assay was conducted accordingto the manufacturer's directions. Briefly, 0.1-10 ng of insulinstandards (100 μl) were added to 12×75 mm test tubes. Similarly, samples(25 μl) from the insulin secretion studies were also added to the testtubes. To this, an aliquot (75 μl) of assay buffer was added. The ¹²⁵Ilabeled insulin (100 μl) was then added to each test tube. An aliquot of100 μL anti rat insulin antibody was added to the tubes, mixed andincubated at 4° C. for 24 h and incubated further with 1 ml aliquot ofthe precipitating reagent for 20 min at 4° C. to precipitate the insulinbound to the antibody. The tubes were then centrifuged and theradioactivity was measured using a gamma counter.

Lowry protein Assay. The amount of protein in the assay wells wasdetermined by Lowry method. The Lowry assay solution was prepared bycombining the Lowry solution, CuSO₄.5H₂O (1%), and sodium tartarate(1%). Briefly, the protein sample (100 μl) and Lowry mixture (1 mL) weremixed in a test tube (12×75). The Folin-Ciolatues reagent (100 μl) wasadded to these tubes, mixed, and incubated for 30 min at roomtemperature. The optical density of resulting solutions was read at 700nm using a UV spectrophotometer.

Results and Discussion

The investigation of Cornus fruits indicated that the primary bioactivecomponents in them were cyanidin, delphinidin and pelargonidinglycosides. Therefore, the attention was focused on the insulinsecreting ability of these anthocyanins and their aglycones usingpancreatic beta cells in order to substantiate the anecdotal use ofCornus fruits in anti-diabetic preparations. Petunidin, malvidin andpeonidin aglycones were included in the assay since they are abundant inother fruits.

Anthocyanins are water-soluble compounds. The aqueous extracts of C. masfruits contained sugars, bioflavonoids and anthocyanins and hence wasfractionated by XAD-16 resin. The resulting anthocyanin fraction elutedfrom the resin was purified by MPLC to afford pure anthocyanins. Theglucose-induced insulin production by INS-1 832/13 cells was determinedat 4, 10 and 16 mM glucose concentrations and found that the insulinsecretion reached a lag phase at 10 mM glucose concentration (data notpresented). The glucose concentration at 4 mM level is representative ofthe normal glucose level in human. The insulin secretion per mg ofprotein by cells at 10 mM glucose was three fold higher when compared tothe insulin secretion at 4 mM glucose concentration.

Anthocyanins and anthocyanidins were tested at 4 and 10 mM glucose loadsin the cell growth medium. Anthocyanins and anthocyanidins were assayedinitially at 50 μg/mL concentration. The anthocyanin, cyanidin3-glucoside showed an increase in insulin secretion at 4 mM glucose by 9ng/mg of protein (1.3 fold) whereas it enhanced the insulin secretion by1.43 fold (119 ng/mg protein) at 10 mM glucose concentration (FIG. 2A).Delphinidin-3-glucoside was the most active anthocyanin tested andshowed a 1.8-fold increase (49 ng/mg of protein) in insulin secretion at4 mM glucose concentration. However, at 10 mM glucose it exhibited onlya 1.4-fold (113 ng) increase (FIG. 2A) in insulin production. Theinsulin secreted by cells at 4 and 10 mM glucose concentrations in thisassay were 27 and 83 ng of insulin per mg protein, respectively. Theanthocyanins, cyanidin-3-galactoside and pelargonidin-3-galactoside, didnot increase the insulin secretion at 4 mM glucose concentration.However, cyanidin-3-galactoside showed an increase of 17 ng/mg ofprotein of insulin (1.2 fold) at 10 mM glucose concentration (FIG. 3).The pelargonidin-3-galactoside was tested only once due to thelimitation of sample.

The anthocyanin cyanidin-3-glucoside was evaluated for dose dependentinsulin secretion at 5, 10, 50, 100 and 250 μg/mL concentrations. Theglucose concentration used in this assay was 4 mM level which isrepresentative of the normal glucose level in human. At thisconcentration, untreated cells secreted 33 ng of insulin/mg of protein.The insulin secreted by cyanidin-3-glucoside treated cells was 46 ng ofinsulin per mg protein at 5 μg/mL. However, there was no significantdifference in insulin secretion at 10, 50, 100 and 250 μg/mLconcentrations of compound 1. There was not an adequate supply ofdelphinidin-3-glucoside to conduct dose dependent assays.

The anthocyanidins were assayed at 50 μg/mL concentration. The aglyconeof cyanidin-3-glucoside, cyanidin, enhanced insulin secretion by 1.5fold (29 ng/mg of protein) at 4 mM glucose whereas at 10 mM glucose itsecreted 88 ng/mg of protein (FIG. 2B). The untreated cells at 4 and 10mM glucose secreted 19 and 83 ng insulin/mg of protein, respectively, inthis set of assay. The aglycone delphinidin showed an increase ininsulin secretion by 6 ng/mg of protein at 4 mM glucose concentrationand was not significant. Delphinidin did not show glucose-inducedinsulin secretion at 10 mM glucose (FIG. 2B). Pelargonidin was the mostactive anthocyanidin and it secreted 49 (1.4 fold) and 91 (1.2 fold) ngof insulin/mg of protein at 4 and 10 mM glucose, respectively (FIG. 3).The aglycone petunidin increased insulin secretion by 4 ng of insulin/mgprotein at 4 mM glucose concentration. However, malvidin did not show anincrease in insulin secretion with respect to the untreated cells.

The results suggested that both anthocyanins and anthocyanidins areinsulin secretagogues. The most potent among them wasdelphinidin-3-glucoside and it significantly induced the insulinsecretion at 4 and 10 mM glucose concentrations compared to theuntreated cells. Although cyanidin-3-glycoside was less active thandelphinidin-3-glucoside at lower glucose concentration, it was moreactive at higher glucose concentration. Among the galactosides,pelargonidin-3-galactoside did not induce insulin secretion at 4 and 10mM glucose concentrations studied, whereas cyanidin-3-galactoside showedsignificant increase in insulin secretion. The ability of anthocyaninsstudied to secrete insulin was in the increasing order ofdelphinidin-3-glucoside>cyanidin-3-glucoside>pelargonidin-3-galactoside.This indicated that the number of hydroxyl groups in ring-B ofanthocyanins played an important role in their ability to secreteinsulin. Among the anthocyanidins tested, pelargonidin was the mostactive at 4 mM glucose. Other aglycones did not potentiate significantinsulin secretion at 4 or 10 mM glucose concentrations studied.

The results suggest that isolated and purified anthocyanins andanthocyanidins from fruits and vegetables are useful to treat diabetes.

Examples of Treatment of Obesity

Anthocyanins (FIG. 9), ursolic and betulinic acids from C. mas fruitswere purified and evaluated their efficacy by using C57BL/6J transgenicmice as agents to prevent obesity and insulin resistance resulting fromthe consumption of high fat diet. The mice were fed initially for fourweeks with high fat diet and were then switched to high fat dietcontaining test compounds for another eight weeks. The glucose tolerancetest (GTT) revealed that the high fat diet control mice were insulinresistant and the mice treated with anthocyanins and ursolic acidovercame the insulin resistance. The average weight gain of control micefed with the high fat diet (60% K. Cal.) during the treatment period was9.76±0.55 g, whereas the mice treated with anthocyanins, betulinic andursolic acids were 7.41±0.93, 7.73±0.44 and 8.78±0.96 g, respectively.The cholesterol levels of the anthocyanins and betulinic acid treatedmice were significantly lower than the control animals. The plasmainsulin levels of anthocyanins and betulinic acid treated animals were567±32.36 and 460.86±93.68 ng/mL, respectively, whereas the animalstreated with ursolic acid showed 52.25±8.84 ng/mL of insulin compared tothe control animals. This in vivo study confirmed that anthocyanins areexcellent insulin secretagogues and may be beneficial in preventingobesity and insulin resistance in addition to lowering the totalcholesterol.

Experimental Procedures

Purification of Anthocyanins: Cyanidin galactoside, pelargonidingalactoside and delphinidin galactoside were isolated as a pure mixtureof anthocyanins from the C. mas fruits as previously disclosed. Briefly,the seeds were separated and the resulting pulp was blended with water(pH=3) and filtered. The filtrate was adsorbed onto XAD-16 AMBERLITEresin and washed repeatedly with water to remove the sugars and otherorganic acids. The adsorbed anthocyanins were then eluted with acidicMeOH (pH=3). The anthocyanins mixture thus obtained was purified bymedium pressure liquid chromatography (MPLC) column (C18 silica) usingMeOH:H₂O (pH=3) under gradient conditions. The fractions eluted withsolvent system MeOH:H₂O (65:35, v/v) were collected and evaporated todryness under vacuum. The purity of anthocyanins was confirmed by HPLC(Waters Corp.) using Capcell C₁₈ analytical column and detected at 520nm (PDA, Waters Corp.).

Isolation of Betulinic acid: The seeds (700 g) from C. mas fruits (5 Kg)were separated, lyophilized and extracted with n-hexane (3×1 L), ethylacetate (3×1 L), and methanol (3×1 L), successively. The EtOAc extract(3.0 g) was purified by silica gel MPLC under gradient conditions withn-hexane and EtOAc as the mobile phases. The fractions collected fromhexane-EtOAc (7:3) elution were evaporated to dryness andcrystallization from MeOH gave betulinic acid (2.5 g).

Isolation of Ursolic acid: The lyophilized pulp and skin were extractedwith n-hexane (3×1 L), EtOAc (3×1 L) and MeOH (3×1 L) successively. TheEtOAc (3.5 g) extract was purified over column chromatography usingn-hexane and EtOAc gradients. The hexane-EtOAc (7:3) eluates wereevaporated to dryness under vacuum and crystallization of the resultingresidue from MeOH yielded ursolic acid (2.2 g). Both ursolic andbetulinic acids were characterized by ¹H and ¹³C NMR spectralexperiments (Werner, S., Nebojsa, S., Robert, W., Robert, S., and Olaf,K. (2003) Complete assignments of ¹H and ¹³C NMR resonances of oleanolicacid, 18α-oleanolic acid, ursolic acid and their 11-oxo derivatives.Mag. Res. Chem. 41, 636-638.).

Animals and Diet: Male C57BL/6J mice, 4 weeks old, were purchased fromJackson Laboratories (Bar Harbor, Me., USA). The mice were individuallyhoused under controlled temperature (70° F.) and 12 h light-dark cycles.The mice (n=40) had free access to water and laboratory non-purifieddiet for 5 days. After acclimatization, the mice were randomly dividedinto groups 1-5 (n=8) for the study. The experiments were carried outaccording to the ethical guidelines of University Laboratory AnimalResources (ULAR) at Michigan State University, East Lansing, Mich. Thediets, 10% K. Cal. (normal) and 60% K. Cal. (high fat), were purchasedfrom Research Diets (New Brunswick, N.J.). The composition of the dietis shown in the Table 1.

TABLE 1 Composition of Normal (105 K. Cal.) and High Fat (60% K. Cal.)Diets Ingredients Normal High fat Casein 200 200 L-Cystein 3 3 CornStarch 315 0 Maltodextrin 35 125 Sucrose 350 68.8 Cellulose 50 50Soybean oil 25 25 Lard 20 245

The controls were groups 1 and 2 and received normal (10% K. Cal.) andhigh fat (60% K. Cal.) diets, respectively, throughout the study. Thefood was prepared for each treatment separately by mixing 1 g of pureanthocyanin mixture, 500 mg each of betulinic and ursolic acids perkilogram high fat diet. The treatment groups, 3-5, were fed initiallyfor 4 weeks with the high fat diet and then switched to the dietcontaining anthocyanins, betulinic acid or ursolic acids. Food waschanged at intervals of three days to avoid oxidation of the fat orcompounds. The daily food intake (FIG. 1) and the weekly body weight foreach animal were determined throughout the study (FIGS. 2A and 2B).

Collection of serum, liver and adipose tissue. The feeding wasterminated after 12 weeks. The animals were then anesthetized by usingisoflurane, sacrificed and blood was collected by cardiac puncture inheparinized tubes. The plasma was separated by centrifugation at 1600×gfor 10 min at 4° C., frozen immediately and stored at −20° C. until use.The liver and epididymal white adipose tissue (WAT) were collectedaccording to the anatomical landmarks, weighed and immediately frozenunder liquid nitrogen. Also, the limb muscles were collected and frozenin liquid nitrogen. The pancreas were collected and stored in optimalcutting temperature (O.C.T) (Sakura Finetek, Inc., CA) and frozen inliquid Nitrogen. All tissues were then transferred from liquid nitrogenand stored at −80° C. until analyses.

Glucose Tolerance Test (GTT). The glucose tolerance test was performedon five animals from each group (n=5) after 6 weeks of supplementation.The blood glucose level was measured at time 0 (min) with a Free StyleFlash (TheraSense, Inc., CA) handheld glucometer using the test strips(Free Style, TheraSense, Inc., CA). For GTT, a sterile solutioncontaining 2 g of glucose per kg body weight was injectedintraperitoneally (i.p.). The tail vein blood was collected and glucoselevels measured at 5, 10, 15, 30, 60, and 90 min, respectively. Theblood glucose levels were plotted against the time (FIG. 3).

Radio Immuno Assay (RIA). The plasma insulin levels were measured by ratinsulin RIA kit purchased from LINCO Research Inc. (St. Charles, Mo.).The insulin standards (100 μl aliquots) were pipetted to 12×75 mm testtubes. A total of 10 concentrations of insulin, ranging from 0.1-10ng/mL, were used to determine the standard curve. The plasma samples(aliquots of 1-25 μl) were added to test tubes and the assay buffer wasadded to attain a total sample volume of 100 μl. The ¹²⁵I labeledinsulin and anti rat insulin antibody (100 μL each) were added to thetubes, mixed and incubated at 4° C. After 24 h, the precipitatingreagent (1 mL) was added and incubated again at 4° C. for 20 min toprecipitate the insulin bound to the antibody. The tubes were thencentrifuged for 20 min at 3000 g, decanted and the radioactivity wasmeasured using a gamma counter.

Determination of Plasma Cholesterol: The total plasma cholesterol wasanalyzed by Clinical Pathology Laboratory at the Diagnostic Center forPopulation and Animal Health, College of Veterinary Medicine, MichiganState University according to the established standard analyticalprotocol for total cholesterol.

Results and Discussion

Cornus mas fruits, also known as cornelian cherry, are similar to tartcherries (P. cerasus). The phytochemical examination of this plantyielded pelargonidin galactoside, cyanidin galactoside, and delphinidingalactoside as the major anthocyanins (Seeram, N. F., Schutzki, R.,Chandra, A., and Nair, M. G. (2002) Characterization, quantification,and bioactivities of anthocyanins in Cornus species. J. Agric. FoodChem, 50, 2519-2523) and triterpenoids such as ursolic and betulinicacids. The transgenic model mouse is regularly employed as a model tostudy the metabolic and endocrine disorders. The C57BL/6J model miceused were homozygous for a leptin receptor mutation and develophyperphagia, obesity, hyperinsulinemia and hyperglycemia (Coleman, D.(1978) Obese and diabetes: two mutant genes causing diabetes-obesitysyndromes in mice. Diabetologia 14, 141-148). Therefore, the mice werefed with the purified anthocyanins, betulinic and ursolic acids from C.mas to evaluate their efficacy in the prevention of diet-induced obesityand insulin resistance. The animals were fed on high fat diet for fourweeks prior to the treatment of compounds, incorporated in high fatdiet, for eight weeks. The control groups of animals received eithernormal or high fat diets.

Body weight and Food Intake. The food intake for the group-1 animals wasaround ≅4.5 g for the first three weeks and then decreased to ≅3.5 g perday (FIG. I) and stayed steady throughout the experiment. The foodintake for group-2 animals was steady throughout the experiment and was≅2.8 g per day (FIG. 1). It is evident from the results that testcompounds did not affect the food intake of the animals. The amount offood intake by animals in groups 3-5 were also about 2.8 g per daythroughout the experiment (FIG. 1).

The body weights of animals in group 1 (normal diet) and 2 (high fatdiet) were significantly different with an average weight of 31.5 and36.91 g, respectively. The animals on high fat diet treated withcompounds 1 (group 3), 2 (group 4) and 3 (group 5) weighed 34.19, 33.54and 34.89 g, respectively (FIGS. 2A and 2B). The overall weight gainduring the experimental period (12 weeks) for the group-1 and -2 animalswere 13.94 and 18.98 g, respectively. Similarly, the group 3, 4, and 5animals gained 15.91, 15.16 and 17.45 g of bodyweight, respectively. Theweight gained by these animals during the treatment period was 7.41,7.73 and 8.78 g, respectively, whereas the group-1 and -2 controlsshowed the bodyweight gain of 6.63 and 9.76 g, respectively.

Glucose Tolerance Test. The glucose tolerance test (GTT) was conductedby intraperitoneal (i.p.) injection of the glucose solution (2 g/kg).The blood glucose levels in animals injected with glucose weredetermined by drawing blood from tail vein at 5, 10, 15, 30, 60 and 90min intervals. The zero time blood glucose level among the groups werealmost identical. The initial glucose levels of low fat and high fatcontrol groups (1 and 2) were 133.8±15.37 and 119.8±7.24 mg/dL,respectively (FIG. 3). The blood glucose level determined in animalstreated with compounds 10-13 were 123.4±4.65, 123.4±6.0 and113.5±15.5.16 mg/dL, respectively. The blood glucose concentrationreached the maximum at 30 min after the glucose injection in all groupsexcept for ursolic acid treated animals. Also, the glucose absorptionwas slow in this group and the blood glucose concentration reached themaximum at 60 min. After 90 min of glucose load, the blood glucoselevels of animals fed with normal and high fat diet were 190±6.31 and363±19.76 mg/dL, respectively. Similarly, the blood glucose levels ofanimals in groups 3-5 were 221±31.5, 317.8±21.9 and 227±22.982,respectively.

Plasma Insulin Levels: The plasma insulin was measured by using RadioImmuno Assay (RIA) (Qian, D., Zhu, Y., and Zhu, Q. (2001) Effect ofalcohol extract of Cornus officinalis Sieb. et Zucc on GLUT4 expressionin skeletal muscle in type 2 (non-insulin-dependent) diabetes mellitusrats. Zhongguo Zhongyao Zazhi 26, 859-862). The insulin levels measuredfor control animals, groups 1 and 2, were 0.47±0.14 and 0.41±0.1 ng/mL,respectively (FIG. 4), where as the animals treated with theanthocyanins, betulinic and ursolic acids showed 567.98±32.36, 460±93.68and 52.25±8.84 ng/mL of insulin, respectively.

Fasting Blood Glucose: The fasting blood glucose of the normal and highfat diet controls were measured to determine whether the animalsconsumed the high fat diet were diabetic or not. The animals weredeprived of food for 6 h and the glucose levels were determined fromblood collected from the tail vein. The glucose levels of normal (n=8)and high fat diet (n=8) fed animals were 126.6±4.6 and 125±5.19 mg/dL,respectively.

Plasma Cholesterol: The plasma cholesterol levels of the normal and highfat diet controls were 120.5±10.61 and 156.4±8.26 mg/dL, respectively.The cholesterol of the anthocyanins and betulinic acids treated animalswas 134.2±15.5 and 126.5±14.01 mg/dL, respectively (FIG. 5).

The food intake of animals on high fat diet alone and high fat dietcontaining test compounds did not vary over the course of the study. Itis interesting to note that the control animals on normal diet consumedmore food than the animals on high fat diet. The caloric intake by thehigh fat diet control and treatment groups was about 14.56 K. Cal. perday whereas the normal diet controls consumed 13.3 K. Cal. per day.

The animals fed on anthocyanin containing diet showed a remarkabledecrease in bodyweight as compared to the high fat diet controls. Theweight loss observed for anthocyanins and betulinic acid fed animalswere 24 and 21%, respectively (FIGS. 5A and 5B). However, the weightloss observed for animals fed on ursolic acid was not significantcompared to the high fat diet control. The plasma of anthocyanin andbetulinic acid treated animals showed a considerable decrease in totalcholesterol compared to the high fat diet control (FIG. 5). The plasmafrom ursolic acid treated animals was not sufficient enough to completethe total cholesterol assay. The food intake for animals in groups 2-5was similar throughout the study and hence the weight loss observed foranthocyanins fed animals suggested its potential application in theprevention of obesity.

Glucose tolerance test (GTT) was carried out on all animals to determinethe insulin resistance (FIG. 6). Even though ursolic acid did notdecrease bodyweight of the treated animals significantly, all animals inthis group corrected glucose levels similar to control group animals fedon normal diet. The anthocyanin treatment showed similar effect as inthe case of ursolic acid treatment in the GTT assay except that theblood glucose concentration reached the maximum at 30 min. The bloodglucose level of the animals treated with ursolic acid (group 5) reachedthe maximum at 60 min indicating that ursolic acid may be delaying theglucose absorption. Therefore, ursolic acid may be a useful product tobe consumed by type-2 diabetic patients since it has the ability todelay the absorption of glucose. At 90 min, the blood glucose levels ofanthocyanin and ursolic acid treated animals were similar to the controlgroup which received the low fat diet. However, the animals treated withbetulinic acid did not respond in GTT and the results were similar tothe control group fed on high fat diet. In the case of high fat diet fedanimals, the blood glucose concentration reached the maximum at 30 minand stayed steady up to 90 min showing that these animals were insulinresistant.

The plasma insulin concentrations of the animals treated with compounds10-15 of FIG. 9 were considerably higher than the control animalsreceived normal and high fat diets (FIG. 4). The increase in insulinsecretion by anthocyanin treated animals was the highest amongtreatments. The insulin secretion by anthocyanin treated animals was 10times or more than the ursolic acid treated animals (FIG. 7). Inconclusion, the anthocyanins isolated from C. mas fruits was the best ofthree compounds studied in reducing the body weight of the animals onhigh fat diet. It also induced the secretion of an enormous amount ofinsulin without causing hypoglycemia.

The methods for the separation of and production of the anthocyanins andanthocyanidins are described in U.S. Pat. Nos. 6,194,469; 6,423,365;6,623,743; 6,676,978 and 6,656,914; and U.S. patent application Ser. No.10/084,575, filed Feb. 27, 2002 which are incorporated by referenceherein in their entireties.

It is intended that the foregoing description be only illustrative ofthe present invention and that the present invention be limited only bythe hereinafter appended claims.

1. A process of increasing insulin secretion in a subject in needthereof comprising administering to the subject a polyphenol activeingredient consisting essentially of cyanidin-3-glycoside alone in apharmaceutically acceptable carrier to increase the insulin secretion.2. The process of claims 1 wherein said polyphenol active ingredient andsaid pharmaceutically acceptable carrier are essentially free of sugars.